Affinity-based detection of biological targets

ABSTRACT

A method of biochemical identification by: providing a plurality of capture species bound to one or more substrates and suspected of having one or more biological targets affinity bound to at least one capture species; detecting which capture species contain bound biological targets to generate a binding pattern; and identifying the biological target based on the binding pattern. The capture species are independently selected from the group consisting of antimicrobial peptides, cytotoxic peptides, antibiotics, and combinations thereof. A device having the capture species bound to the substrates. At least two of the capture species are capable of multi-specific binding to one or more biological targets and may have overlapping but not identical affinity properties.

This application is a divisional application of allowed U.S. patentapplication Ser. No. 11/307,399 filed on Feb. 6, 2006, which claimspriority to U.S. Provisional Patent Application No. 60/690,046, filed onJun. 10, 2005. The prior applications and all other publications andpatent documents referred to throughout this nonprovisional applicationare incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is generally related to biological detection.

2. Description of Related Art

With the notable exception of glucose sensors, the vast majority ofrapid detection/measurement systems use antibodies for recognition,identification, and quantification of biological targets. Antibody-baseddetection techniques are powerful, versatile tools for various molecularand cellular analyses, environmental monitoring, and clinicaldiagnostics. This power originates from the specificity of theantibodies for their particular antigenic sites.

Antibody-based recognition of targets is the basis for detection in manyoptical and electrochemical biosensors (e.g., interferometers,reflectometric interference spectoscopic sensors, resonance minorsensors, surface plasmon resonance instruments, quartz crystalmicrobalances, light-addressable potentiometric sensors,electrochemiluminescence systems, fiber optic, and array biosensors), aswell as in flow cytometry and non-sensor detection techniques such aslateral flow assays.

Detection techniques employing antibodies, although considered lesssensitive than polymerase chain reaction-based systems, are still highlysensitive, are well characterized, and have been adapted for use inrapid assay systems. Due to the specificity of the antibodies, many ofthese immunoassay-based systems have the additional benefit of requiringlittle if any sample preparation prior to analysis.

However, assays utilizing antibodies for specific recognition of targetanalytes have a number of problems that may significantly limit theirwidespread use in the field: 1) many antibodies are sensitive toenvironmental temperatures and must be stored frozen, refrigerated, orlyophilized for retention of optimal activity; 2) at least one antibodyor set of antibodies is required for each target of interest inmultiplexed assays, increasing the complexity and potential fornon-specific or cross-reactive binding; 3) specificity and sensitivityof antibody-based recognition may, in some cases, be mutually exclusive;4) target-specific antibodies may not be available due to thenon-antigenic nature of the analyte; and 5) although monoclonalantibodies are, by their very nature, more consistent than polyclonalantibodies, development and large-scale production of monoclonals isexpensive and time-consuming.

The biological detection and clinical diagnostic markets are currentlydominated by antibody-based assays. However, antibody-based assays maynever be stable enough for long-term sensor applications; such stabilityis critical for fielding sentry-type systems and for non-laboratory use.Use of antimicrobial peptides and antibiotics should improve the currentlogistical burdens required of fielded systems.

Many organisms, including mammals, insects, amphibians, fish,crustaceans, plants and bacteria, produce antibiotics and antimicrobialpeptides as part of their innate immune systems for protection againstinvasion by harmful microbes. Antimicrobial peptides and someantibiotics recognize target pathogens by interacting with the microbialcell membranes. Most peptide-membrane and antibiotic-membraneinteractions do not involve specific receptors, but rather invariantcomponents of the cell surface; binding is therefore semi-selective—eachpeptide or antibiotic can bind to multiple microbial species withdiffering affinities. As natural evolution has conferred upon many ofthese compounds the stability to withstand adverse conditions (pollutedponds, etc.) and the ability to recognize multiple microbial species,assays using these peptides and antibiotics for recognition should havethe following advantages over conventional antibody-based screeningmethods: stability, resistance to proteases, ability to detect largernumbers of species than a corresponding number of antibodies, and alower degree of complexity for multi-analyte screening assays.

SUMMARY OF THE INVENTION

The invention comprises a biochemical identification method comprising:providing plurality of capture species bound to one or more substratessuspected of having one or more biological targets affinity bound to atleast one capture species; detecting which capture species contain boundbiological targets to generate a binding pattern; and identifying thebiological target based on the binding pattern. The capture species areindependently selected from the group consisting of antimicrobialpeptides, cytotoxic peptides, antibiotics, and combinations thereof.

The invention further comprises a device comprising: one or moresubstrates; and a plurality of capture species bound to the substrates.The capture species are independently selected from the group consistingof antimicrobial peptides, cytotoxic peptides, antibiotics, andcombinations thereof. At least two of the capture species are capable ofmulti-specific binding to one or more biological targets.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Example Embodiments andthe accompanying drawings.

FIG. 1 shows the effect of fluorophore on non-specific binding insandwich assays with non-specific binding of Cy5 labeled antibodies

FIG. 2 shows the effect of fluorophore on non-specific binding insandwich assays in detection of E. Coli and Salmonella on a multipeptidearray with Cy3 labeled antibodies.

FIG. 3 shows different binding patterns of E. coli and Salmonella tovarious peptides and antibiotics—Luminex¹⁰⁰.

FIG. 4 shows different binding patterns of E. coli and Salmonella onvarious peptides and antibiotics—Array Biosensor.

FIG. 5 shows detection of a toxin using multiplexed antibiotics andantimicrobial peptides. Botulinum toxoid bound to Cecropin A and (lessstrongly) to polymyxin B, but not to magainin.

FIG. 6 schematically illustrates immobilization of AMPs on a glass slidefollowed by binding of a fluorescent target.

FIG. 7 shows charge-coupled device images of Cy5-labeled cells bindingto immobilized magainin on sensing arrays.

FIG. 8 shows concentration-dependence curves for Cy5-labeled Salmonella(A) and E. coli (B) cell binding to magainin immobilized by directattachment through cross-linking chemistry with GMBS (◯) and byavidin-biotin chemistry (▪).

FIG. 9 shows binding of Cy5-labeled Salmonella (1×10⁷ cell/mL) todifferent densities of C-terminal biotinylated magainin.

FIG. 10 shows formats for Luminex based detection.

FIG. 11 shows detection of E. coli and Salmonella—Luminex. Capture usingAMP-derivatized Lx beads; antibody tracers.

FIG. 12 shows patterns of binding by E. coli (10⁶ cfu/ml), E. faecalis(10⁴ cfu/ml), and B. fragilis (10⁵ cells/ml), in Luminex AMP-tracerassays. Values normalized to colistin signals.

FIG. 13 shows binding patterns for Luminex AMP capture—E. coli,Salmonella (10⁶ cfu/ml each), bot toxoid A (10 μg/ml).

FIG. 14 shows binding patterns for Array Biosensor—E. coli, Salmonella(10⁶ cfu/ml each), C. burnetti (2×10⁶ cells/ml), Brucella (5×10⁴cfu/ml).

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

In the following description, for purposes of explanation and notlimitation, specific details are set forth in order to provide athorough understanding of the present invention. However, it will beapparent to one skilled in the art that the present invention may bepracticed in other embodiments that depart from these specific details.In other instances, detailed descriptions of well-known methods anddevices are omitted so as to not obscure the description of the presentinvention with unnecessary detail.

The invention may use multiple antimicrobial peptides or otherantibiotics as recognition molecules for detection of toxins, viruses,bacteria, rickettsiae, and fungi. The antimicrobial peptides andantibiotics can be used in any detection system in place of antibodies(or other specific receptors) as “capture” species or “tracer” species,as appropriate. A key concept is the use of multiple antimicrobialpeptides or antibiotics for target recognition. Based on theiroverlapping specificities for various targets, a database of differentbinding patterns can be developed; targets can then be identified basedon differences in responses by use of a pattern recognition algorithm.The semi-selectivity of binding can allow detection of a larger numberof targets than a corresponding number of antibodies or target-specificreceptors. Antimicrobial peptide- and antibiotic-based assays mayincrease the multi-analyte detection capabilities and improve logisticrequirements of current antibody-based detection systems and be amenableto use in any detection platform that currently utilizes antibodies fortarget recognition.

The method and/or device described herein may differ from othermultiplexed peptide- and antibiotic-based detection systems in one ormore of the following aspects:

-   1. The capture species are not combinatorially derived or randomly    generated. Rather, they are peptides or antibiotics found in nature,    whose binding properties to microbial surfaces (and in some cases,    to toxins) may be documented. The capture species are not limited to    only naturally occurring anti-microbial peptides and antibiotics,    but may also include chimeric peptides, genetic variants, and    synthetic mimics.-   2. The arrayed capture species have defined secondary structures,    unlike combinatorially derived or randomly generated libraries of    peptides, aptamers, sugars, etc.-   3. Binding of the multiplexed capture species is used to detect    bacteria, viruses, fungi, rickettsial targets, and toxins. The    detection mechanism is affinity-based, not enzymatically based.-   4. The antimicrobial capture species used in the arrays have    overlapping specificities. Due to the semi-selective binding    characteristics of these molecules, smaller numbers of peptides may    be used to detect larger numbers of targets by use of a pattern    recognition algorithm to deconvolute data for target identification    by these molecules.

Antimicrobial peptides (AMPs) are part of a host's innate immune systemin many organisms and serve as the first line of defense againstmicrobial invasion. Highly stable to adverse conditions, AMPs bindsemi-selectively to microbial cell surfaces and exert theirantimicrobial activity through membrane disruption. Given their abilityto bind to multiple target microbes, an array consisting of multipleAMPs can potentially be capable of detecting a higher number of targetspecies than an array with a corresponding number of antibodies.Furthermore, the predicted stability of the AMPs within these arrays isexpected to improve operational and logistical constraints over currentantibody-based systems. The AMP-based arrays differ from standardpeptide arrays in that some or all components are naturally occurring(or derivatives of molecules produced in nature) and may have definedsecondary structures, unlike combinatorially derived libraries. Mostimportantly, as many AMPs have overlapping specificities, the pattern ofdifferences in binding affinities can be used for identification.

One class of AMPs is comprised of linear peptides that naturally fold toform two helical domains: a strongly basic helical region and ahydrophobic helix separated by a short hinge region. Magainins and otheramphipathic α-helical AMPs are unstructured in solution, but becomehelical upon interaction with target membranes. Because of its stabilityand ability to bind to multiple bacterial species magainin I(GIGKFLHSAGKFGKAFVGEIMKS (SEQ ID NO 12)) may be used as a recognitionmolecule for incorporation into an array-based sensor for detection ofpathogenic bacteria.

In general, use of multiplexed antimicrobial peptides and antibioticsfor target detection may possess the following advantages over standardantibody-based detection techniques:

-   1. Equivalent or superior detection of target analytes. This has    already been demonstrated with botulinum toxoid, but will depend on    the binding constants of each recognition molecule for the targets.-   2. Superior storage characteristics. Antibody-based and DNA-based    detection systems are logistically burdensome. As many antimicrobial    peptides and antibiotics have evolved for survival in active form in    the environment, it is anticipated that activity will be retained    under harsher conditions than can be used with conventional    antibody-based techniques-   3. Capability for simultaneous detection and identification of a    larger number of targets than a corresponding number of antibodies    or nucleic acid probes. Due to the overlapping, semi-selective    binding characteristics of the peptides and antibiotics, fewer of    these recognition molecules will be needed to detect large numbers    of targets.-   4. Potential for therapeutics and/or decontamination. The pattern of    binding may be indicative of sensitivity of target bacteria,    rickettsiae, and fungi to various antibiotics and antimicrobial    peptides.

Since the invention does not rely on a single transduction mechanism(e.g., amperometric detection of an enzymatic product), the inventionmay be adaptable from fluorescence to other detection systems. Theinvention may use antimicrobial peptides and antibiotics for eithercapture and tracer elements or both. The multiplexed antibiotics and/orpeptides can also be used in conjunction with antibodies and otherrecognition species in either orientation.

Factors that may lead to optimal performance include but are not limitedto: 1) direct immobilization of capture peptides/antibiotics onto sensorsubstrates; 2) use of selected fluorophores; and 3) use of a second beadwhen using peptides/antibiotics as tracers. It is possible that thesefactors may vary depending on the detection platform used with themultiplexed antimicrobial peptides and antibiotics.

The device and method may be used with a variety of capture species andbiological targets. Suitable biological targets include, but are notlimited to, bacteria, fungi, viruses, rickettsiae, toxins, andcombinations thereof. Suitable capture species include, but are notlimited to, alamethicin, peptaibols, apidaecin, bacitracin, bactenecins,bombinin, brevinin, buforins, cathelicidins, cecropins,cepaphalosporins, cytolysins, dermaseptins, defensins, esculentins,gramicidins, hemolysins, histatin, indolicidins, beta-lactams,lactoferricin, nisin, lantibiotics, magainins, mastoparans, melittin,moricin, parasin, pediocin, penicillins, polymyxins, protegrins,ranalexin, streptogamins, tachyplesins, teichoplanin, thionins,vancomycin, vibriolysins, derivatives thereof, and combinations thereof.Any number of the capture species may be naturally occurring peptides.For example, one, a majority, or all of the capture species may benaturally occurring peptides.

The capture species may be part of an innate immune system providingchemical immunity. They may have, but are not limited to, 12-45 aminoacids. They may bind to components of a microbe's surface to disrupt tothe membrane. This may require multiple peptides interacting with themembrane and require peptide-peptide interactions. They may usesemi-selective binding in that the peptide-surface interaction may occuracross different genera, but the strength of interaction variesaccording to the membrane composition and presence or absence ofdifferent membrane components.

Not all interactions between AMPs and membranes of target organisms arefully characterized, but they have been demonstrated to occur in theabsence of specific receptors. Cationic peptides are thought topreferentially interact with negatively charged phospholipids onbacterial and fungal membranes, with only marginal activity againstzwitterionic phospholipids. Most cationic peptides therefore exhibitselective toxicity for bacterial, fungal, and protozoan targets, ratherthan mammalian ones, and may preferentially interact with Gram-negativebacteria over Gram-positive species. On the other hand, AMPs withhydrophobic segments (e.g., melittin, alamethicin) are highly toxic tomammalian cells but also bind with high affinity to bacterial membranes.Table 1 shows some example AMPs.

TABLE 1 Sequences and structures of select antimicrobial peptides (AMPs)Non-ribosomally synthesized AlamethicinAc-UPUAUAQUVUGLUPVUUEQF-OH (SEQ ID NO: 1) U = methylalanine Polymyxin B

fa = fatty acid B = diaminobutyrate Polymyxin E (colistin)

fa = fatty acid B = diaminobutyrate Gramicidin Afo-XGALAVVVWLWLWLW-Et (SEQ ID NO: 4) fo = formyl X = V,I Et =ethanolamine Bacitracin

O = ornithine Amphipathic α-helical Buforin IITRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 6) Cecropin AKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-CONH₂ (SEQ ID NO: 7) Cecropin BKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL (SEQ ID NO: 8) Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (SEQ ID NO: 9) CecA/Mel hybridKWKLFKKIGIGAVLKVLTTG-CONH₂ (SEQ ID NO: 10) DermaseptinALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ (SEQ ID NO: 11) Magainin IGIGKFLHSAGKFGKAFVGEIMKS (SEQ ID NO: 12) Magainin IIGIGKFLHSAKKFGKAFVGEIMNS-CONH₂ (SEQ ID NO: 13) MelittinGIGAVLKVLTTGLPALISWIKRKRQQ-CONH₂ (SEQ ID NO: 14) β-Sheet/β-loopBactenecin (intramolecular disulfides)

Nisin (intramolecular thioethers)

U = 2,3-didehydroalanine X = 2,3-didehydrobutyrine Z = α-aminobutyrateDefensin HNP-1 (intramolecular disulfides)

The mechanism of membrane disruption is believed to occur by formationof either “carpets” or channels. The “carpet” mechanism involves bindingof charged (typically cationic) amino acids to headgroups of membranephospholipids or lipopolysaccharide. After initial binding, AMPsaggregate to form a “carpet,” with helices or β-sheets oriented parallelto the membrane surface. Upon rotation of the AMP chains, hydrophobicside chains are inserted into the membrane, disrupting lipid packing, oralternatively, creating a toroidal pore. Channel formation, on the otherhand, involves insertion of the peptide backbone into the membrane,rather than the side chains. After insertion of the peptide backboneinto the membrane, AMPs aggregate to form a barrel-like structure with acentral aqueous channel. A feature of both mechanisms is the requirementfor multiple AMPs and for peptide-peptide interactions. The attachedclaims are not intended as requiring these or any other mechanisms.

Not every species on the substrates is required to be a capture species.A device may be an array having additional species, including but notlimited to, antibodies, for simultaneously performing other types ofassays. In a device, there are at least two capture species that arecapable of multi-specific binding. There may also be 3, 4, 5, 10, 20,50, or 100 or more such capture species. Optionally, at least two of thecapture species have overlapping, but not identical affinity properties.

The biological target or targets may be bound to the capture species byexposing the substrate or substrates to a sample suspected of containingthe biological target and allowing the target to bind to the capturespecies. In one embodiment, the biological target may be directlydetected by a reagent-less assay. Such assays include, but are notlimited to, opto-electronics, surface plasmon resonance, interferometry,and quartz crystalline microbalance. A reagent may also not be neededwhen the target has a label attached to the target that is capable ofproducing an opto-electronic signal.

In another embodiment, the presence of the biological target is detectedby use of a tracer species that comprises a recognition element capableof binding to the biological target and a signal generating element. Thesubstrate is exposed to one or more such tracer species, which areallowed to bind to the bound biological target. Detecting the biologicaltarget is done by detecting the tracer species bound to it. The devicemay include a reservoir or source of the tracer species. Alternatively,the tracer species may be bound to the biological target before thetarget is bound to the capture species.

Among other possibilities, the signal generating element may be capableof producing an opto-electronic signal, such as fluorescence. Suitablesignal generating elements include, but are not limited to,fluorophores, chromophores, fluorophore-labeled species,chromophore-labeled species, fluorescent nanospheres or microspheres, anenzyme or catalyst capable of producing an opto-electronic signal, andfluorescent nanospheres or microspheres coated with one of the capturespecies. Suitable fluorophore labels include, but are not limited to,Cy3, Cy5, cyanine dyes, phycobili proteins, and fluorescent protein. Thetracer species may also be a fluorescent nanosphere or microspherecoated with a capture species, particularly when used with the Luminexsystem. As used herein, the terms “nanosphere” and “microsphere” aredefined as used anywhere in the relevant art, as opposed to definingstrict dimensions. The tracer species may also be a stain applied to oneor more biological targets, either before or after binding to thecapture species. Such staining assays are described in Ligler et al.,U.S. Pat. No. 5,496,700.

In one embodiment, the substrate may be any flat surface, such as thoseused in the microarray art, including but not limited to, a glass slide.The surface of the substrate may be functionalized with a crosslinker,with the capture species covalently bound to the crosslinker.Alternatively, the capture species may be non-covalently bound to thesubstrate, or may be bound to a carrier protein or scaffold, which iscovalently or non-covalently bound to the substrate.

In this embodiment, the different capture species may be bound inseparate regions of the substrate. When a biological target or tracerspecies is detected at a particular place on the substrate, it can bedetermined in which region the target is located, and thus, to whichcapture species the target is bound. The regions need not be discrete ordisjoint, as long as it can be determined on which capture species adetection event is located.

The NRL array biosensor generally uses sandwich fluoroimmunoassaysperformed on the surface of an optical waveguide (microscope slide) todetect targets of interest. Typically, biotin-labeled “capture”antibodies are immobilized in a patterned array on an avidin-coatedslide. After sample is flowed over the array, bound target is detectedwith a fluorescently labeled “tracer” antibody, whose presence andlocation are determined using a camera system.

This embodiment of the invention may use an array of multipleantimicrobial peptides and antibiotics. Typical peptide/antibioticimmobilization procedures include (but are not limited to) the followingsteps:

-   -   1. Cleaning of microscope slides using standard methods (KOH in        alcohol or H₂SO₄)    -   2. Silanization with amino-functional or thiol-functional silane    -   3. Treatment with homo- or hetero-bifunctional crosslinker (BS³,        GMBS)    -   4. Placement of a patterning template onto the treated surface    -   5. Overnight incubation with 1-10 mg/mL peptide/antibiotic in        buffer    -   6. Washing of the surface, followed by blocking, drying and        storage

Direct assays were performed by flowing fluorescently labeled target(cells, toxins, etc.) over the surface of the slide and washing awayunbound target. Sandwich assays were performed by flowing (unlabeled)sample over the slide, washing, flowing over a fluorescent tracerantibody, and washing a second time; antibiotic or peptide can also beused as a tracer molecule.

In another embodiment, a plurality of microspherical substrates is used,such as in the Luminex system. There are subsets of microspherescomprises a different capture species or combinations there of bound itsthe surface. Each microsphere is encoded by two dyes. The ratio of thedyes is determined by the identity of the capture species.

The Luminex¹⁰⁰ is a commercial flow cytometer that performs sandwichimmunoassays on the surface of microspheres encoded by different ratiosof two long wavelength dyes. Up to 100 simultaneous assays can beperformed, as Luminex can distinguish between the different bead types.The current embodiment using antimicrobial peptides and antibiotics mayrequire a dual-bead assay for target detection.

In a typical assay, antibody-coated Luminex beads (antibody immobilizedby avidin-biotin interactions) are added to the sample containing thetarget species; these beads could potentially be coated with antibioticsor antimicrobial peptides. After the target has bound to the capturebeads, fluorescent nanospheres coated with antimicrobial peptide areadded to the sample. After a short incubation, the sample is thencentrifuged to pellet both the nanospheres and the microspheres. Thebeads are then resuspended by a short exposure in an ultrasonic bath andthen sample is directly measured by the Luminex flow cytometer.

For both systems, it was found that the antimicrobialpeptides/antibiotics may bind targets more efficiently if they arecovalently attached to a surface. This is in contrast to manyantibody-based detection systems that allow immobilization viaavidin-biotin interaction. Although the immobilization chemistry used inthese examples provides higher levels of binding, the method ofimmobilization may be modified as appropriate for the detectionplatform. Furthermore, the peptides and antibiotics can be attached tobeads, proteins, dendrimers, etc. for capture and detection of targetanalytes.

Characteristics of surface chemistry for the immobilization typicallyconsidered as disadvantages in other systems (e.g., lack of diffusion,steric hindrance) were advantageous when immobilizing the peptides andantibiotics. Tight control over orientation of the molecule on thesurface and prevention of over-labeling may be significant when usingantimicrobial peptides and antibiotics. Furthermore, due to therequirement for strong target interactions, valency (density on thesurface) may also be relevant in endowing peptide-based systems withsufficient strength to capture the target species. This may also explainwhy high molar quantities of peptides were required in theimmobilization step in the examples (0.2-2 mM versus <1 μM typicallyused with antibodies) and why avidin-biotin-based immobilization may notbe sufficient for tight binding.

For the examples discussed herein, the amount of non-specific binding bytracer species was affected by the fluorophore used. Cy5- or AlexaFluor647-labeled tracer species bind non-specifically to immobilized peptidesand antibiotics, regardless of the presence of target analyte (FIG. 1,PBSTB lanes). However, it was determined that tracers labeled with Cy3dye (in which the alkene chain is two carbon units shorter) do not bindnon-specifically (FIG. 2, PBSTB lanes); it was successfully demonstratedthat sandwich assays using Cy3-labeled tracers can be used to detectmultiple targets. The use of Cy3 for successful sandwich assays wassurprisingly advantageous, given the similarity in structure of thedyes. Although this dye was suitable for successful sandwich assaysusing the NRL array sensor, other systems may require differentfluorophores and additional optimization.

A detector may be used to independently detect the presence of thetracer species or the biological target in each region to generate abinding pattern, such as those shown in FIGS. 12-14. The type ofdetector used is dependent on the type of signal generated by the targetor tracer. The pattern describes to which capture species, and to whatdegree, the biological target or targets has bound. The pattern may thenbe compared to a database of known binding patterns to identify thebiological target. The identification may be done by performing apattern recognition algorithm using a database of at least one of thebiological targets characterizing the biological target by its relativebinding affinity for at least one of the capture species. This algorithmmay be performed with a system such as a computer.

The invention can be adaptable to multiple detection platforms,including biosensors. Bacteria were successfully detected on NRL's arraybiosensor and the Luminex¹⁰⁰. Species-specific binding patterns of setsof peptides using both Luminex (FIG. 3) and the NRL array sensor (FIG.4) were demonstrated. These results further demonstrate theproof-of-principle that the pattern of binding can be used todistinguish different targets.

It was also demonstrated that the invention can be used for detection ofnon-bacterial targets. FIG. 5 shows detection of botulinum toxoid Ausing various immobilized antimicrobial peptides and antibiotics, aswell as an appropriate anti-botulinum antibody. The estimated limit ofdetection observed in the peptide-based assays (specifically, in thececropin lane) was significantly lower than previously observed forantibody-based assays. This is also evident in the higher signals shownin FIG. 5. Although inhibition of botulinum toxin by buforin has beenreported (Garcia et al., “Buforin I, a natural peptide, inhibitsbotulinum neurotoxin B activity in vitro,” J. Appl. Toxicol., 19(S1),S19-S22 (1999)), the use of other antimicrobial peptides and antibioticsfor toxin detection was not obvious; their typical mode of action andinteractions is by membrane interaction. These results demonstrate thatthe invention may have additional uses in detection of other(non-microbial) targets.

At least a 10-fold improvement in detection limit was observed overprior art for detection of botulinum toxoid A. Various targets(Salmonella, E. coli) were distinguished based upon their pattern ofbinding to different peptides. It is anticipated that the pattern ofbinding can be used to identify significantly more bacterial (andpotentially fungal, viral, and toxic) targets.

The invention can allow sensitive detection of bacterial cells or cellfragments in a rapid biosensor assay using antimicrobial peptides fortarget recognition. Furthermore, this study demonstratesproof-of-concept that these simple 70-minute AMP-based assays providedsimilar detection limits but greater stability at room temperature thananalogous antibody-based assays. These assays can enable creating amultiplexed detection platform that uses the semi-selective binding ofmultiple AMPs to detect large numbers of bacterial species. Preliminaryevidence that the directly immobilized magainin shows semi-selectivebinding characteristics; only trace binding of Campylobacter sp. andBacillus sp. was observed under analogous conditions.

Having described the invention, the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention describedin this application.

EXAMPLE 1

Array Biosensor—Immobilization of peptides and antibodies—FIG. 6schematically illustrates immobilization of AMPs on a glass slidefollowed by binding of a fluorescent target. The direct method hasattachment of recognition molecules to the substrate throughcross-linking chemistry with GMBS. Indirect immobilization hasattachment of recognition molecules to the substrate through anavidin-biotin interaction. The immobilization methodology utilizessequential incubations with a thiol-terminated silane, theheterobifunctional crosslinker N-[γ-maleimidobutyryloxy]-succinimideester (GMBS), and a protein or peptide containing one or more primaryamines. Briefly, standard soda-lime microscope slides (Daigger, VernonHills, Ill.) cleaned with 10% KOH (w/v) in methanol were treated for 1hour, under nitrogen, with a 2% solution of 3-mercaptopropyltrimethoxysilane in toluene. The slides were then rinsed with toluene,dried under nitrogen, and incubated for 30 minutes in 1 mM GMBScrosslinker (Pierce, Rockford, Ill.) in absolute ethanol. Following thecrosslinker incubation, magainin I and control antibodies wereimmobilized on the surface using either 1) indirect attachment viaavidin-biotin interactions (covalent attachment of an avidin derivative,followed by incubation with biotinylated magainin or antibody), or 2)direct covalent attachment chemistry (interaction of succinimidyl esterwith primary amines on the antibody or magainin).

For avidin-biotin-mediated attachment of capture molecules, the slideswere removed from crosslinker, rinsed briefly in water, and thenincubated overnight in 33 μg/mL NeutrAvidin (Pierce, Rockford, Ill.) inphosphate buffered saline, pH 7.4 (PBS). The NeutrAvidin-treated slideswere rinsed in PBS and stored at 4° C. in PBS until patterned withbiotinylated capture species. Patterning of capture species ontoNeutrAvidin-coated slides was performed by placing a 6-channelpoly(dimethylsiloxane) (PDMS) patterning template onto the surface ofeach slide and filling each channel with an appropriate biotinylatedcapture molecule in PBS. Following overnight incubation at 4° C., eachchannel was emptied and rinsed with PBS. Slides were then blocked for 30minutes in 10 mg/mL gelatin, rinsed with nanopure water, and dried undernitrogen. The following capture species were patterned onNeutrAvidin-coated slides: (1) 1 mg/mL (0.4 mM) custom synthesizedmagainin I possessing a C-terminal biotin; (2) 1 mg/mL (0.4 mM) magaininI labeled with an amine-specific biotin derivative; and (3) 10 μg/mL (66nM) biotinylated control antibodies.

For direct covalent attachment of recognition molecules, slides wereremoved from crosslinker, rinsed briefly in water, dried, and thenplaced in contact with the PDMS patterning templates. Unlabeledantibodies (10 μg/mL in PBS) and magainin I (1 mg/mL in PBS) wereinjected into appropriate channels and incubated overnight at 4° C. Thechannels were then emptied and rinsed with PBS. Patterned slides wereblocked with 10 mg/mL gelatin as above, dried, and stored at 4° C. forup to 2 weeks.

Biotinylation of capture molecules—Rabbit anti-E. coli 0157:H7 (KPL,Gaithersburg, Md.), anti-S. typhimurium (Biodesign, Saco, Me.), andanti-Listeria (Biodesign, Saco, Me.) were biotinylated with a 5-foldmolar excess of the long-chain derivative of biotinN-hydroxysuccinimidyl ester (EZ-Link NHS-LC-biotin, Pierce) according tothe following procedure.

-   -   1. Calculate the amount of biotin-LC-NHS ester needed to for 5:1        (biotin:antibody) ratio.    -   2. Dilute antibody solution such that the final concentration is        1-2 mg/ml. (As the reaction chemistry links biotin (or Cy5) to        the antibody via amine groups, the antibody must be in a buffer        that does not contain moieties. Amine-based buffers (e.g., Tris,        glycine) must be removed from the antibody prep prior to the        labeling reaction. Any method suitable for desalting may be used        for buffer exchange.)    -   3. Add 1/9 volume of 0.5 M bicarbonate buffer, pH 8.5 to the        antibody solution such that the final bicarbonate buffer is 50        mM.    -   4. Dissolve biotin-LC-NHS ester in DMSO to a final concentration        of 1 mg/ml.    -   5. Add the biotin/DMSO mix to the diluted antibody in        bicarbonate buffer, such that the final biotin to antibody ratio        is 5:1.    -   6. Incubate the antibody/biotin mix at room temperature for 30        minutes while rocking.    -   7. Pipette mix onto a Bio-Gel P-10 column, which has been        pre-equilibrated in PBS (The Bio-Gel P-10 column is prepared by        first suspending Bio-Gel P-10 gel (medium, 90-180 μm) into PBS        to make a slurry. To completely hydrate the Bio-Gel, allow the        slurry to sit overnight at room temperature or alternatively,        autoclave or boil for 20 minutes. The hydrated slurry is stable        at room temperature for months. Immediately prior to loading the        column, the slurry is degassed by application of a vacuum. The        slurry is then loaded into a 25 ml column, pre-filled with        approx. 3 ml PBS. Once the column has been filled, it is then        flushed with at least three volumes of PBS. After the elution of        the conjugate products, the column must be flushed exhaustively        with PBS and stored wet (PBS) at room temperature for future        used.). Allow the sample to soak into gel. Rinse top of gel and        sides of column with PBS.    -   8. Add a layer of 1-5 ml PBS onto the top of gel and monitor the        absorbance of the eluent at 280 nm. Collect all eluent by        fractions and save the first peak. Add more PBS buffer as        necessary    -   9. Dilute an aliquot of the first peak fraction with PBS        (typically 10×dilution) and measure the absorbance at 280 nm.    -   10. Determine the concentration of the biotinlylated conjugate        and store at 4° C. The absorbance at 280 nm should be below a        value of 1.0 absorbance units for accurate determination of        concentration. The concentration of biotinylated antibody is        determined by the Beer Lambert Law (A=εcl), with        ε_(280 nm, 1 mg/ml, 1 cm)=1.4.

Unlabeled magainin I (GIGKFLHSAGKFGKAFVGEIMKS (SEQ ID NO 12), AnaSpec,San Jose, Calif.) was incubated with EZ-Link NHS-LC-biotin at a 1:1(biotin:peptide) molar ratio in PBS for 24 hours at room temperature;the biotin was first dissolved in a small volume of dimethylsulfoxide(DMSO) prior to adding to the labeling mix. After 24 hours incubation,samples were loaded into dialysis tubing (1000 MWCO) and dialyzedagainst PBS over 3 days, with 6 changes of buffer. The biotinylatedmagainin I was characterized by electrospray mass spectrometry using aQSTAR pulsar I (Applied Biosystems, Foster City Calif.) with nano-flowdirect infusion. A custom-synthesized magainin possessing a C-terminalbiotin (99% pure, purchased from SynPep, Dublin, Calif.) was also usedin this study.

Preparation of fluorescent cells—Heat-killed S. typhimurium and E. coliO157:H7 cells (KPL) were rehydrated in PBS as recommended by themanufacturer. Approximately 10⁸ cells/mL were incubated for 30 min in 50mM sodium borate, pH 8.5, with one packet of Cy5 bisfunctionalN-hydroxysuccinimidyl ester (Amersham, Arlington Heights, Ill.)dissolved in 25 μL anhydrous DMSO immediately before use. Labeled cellsand unincorporated dye were loaded into dialysis tubing (1000 MWCO) anddialyzed overnight at 4° C. against 3 changes of PBS. Labeled cells werethen removed from the bag and stored in the dark at 4° C. until use.

Assay Protocol—Patterned slides were placed in contact with PDMS assaytemplates molded to contain 6 channels oriented orthogonal to thechannels in the patterning templates. The slides with the attached assaytemplates were connected to a multichannel peristaltic pump at one endof each flow channel via syringe needles (outlet). The opposite end ofeach flow channel was connected to a 1 mL syringe barrel used asreservoir. To rehydrate the slide, each channel was washed with 1 mL ofPBS containing 1 mg/mL bovine serum albumin and 0.05% Tween-20 (PBSTB)at 0.8 mL/min. Samples (0.1 mL Cy5-labeled cells in PBSTB) were theninjected into appropriate channels and allowed to incubate for 1 hr atroom temperature in the dark. Each channel was then washed with 1 mL ofPBSTB at 0.3 mL/min. After removing the PDMS templates, the slides werewashed with deionized water, dried under nitrogen, and imaged using thearray biosensor.

Fluorescence imaging, data acquisition and analysis—Optical componentsof the Naval Research Laboratory's (NRL) array biosensor have beendescribed in Feldstein et al., “Array Biosensor: Optical and FluidicsSystems,” J. Biomed. Microdevices 1(2), 139-153 (1999) and Golden etal., “A comparison of imaging methods for use in an array biosensor,”Biosens. Bioelectron. 17(9), 719-725 (2002). Briefly, it consists of a635 nm, 12 mW diode laser for evanescent excitation of surface-boundfluorophores, a waveguide support, a GRIN lens array, several emissionfilters, and a Peltier-cooled charge-coupled device (CCD) imaging array.Digital images of the pattern of fluorescent spots were captured by theCCD and saved in Flexible Image Transport System (FTS) format. A customdata analysis software program (Sapsford et al., “Kinetics of AntigenBinding to Arrays of Antibodies in Different Sized Spots,” Anal. Chem.,73(22), 5518-5524 (2001)) was used to extract data from the FTS file,calculate the mean fluorescence intensity within each array element, andsubtract out localized background, resulting in a mean net fluorescencevalue for each array element. Limits of detection (LODs) were defined asthe lowest concentration tested for which the mean net fluorescencevalues (n>3) are greater than three standard deviations above bothnegative control values and localized background values.

Fluorescently labeled, heat-killed S. typhimurium (FIG. 2) and E. coliO157:H7 were both detected on arrays where magainin I was immobilizedvia its C-terminal biotin and unmodified magainin I was immobilized bydirect covalent attachment. In general, slides with directly immobilizedmagainin (FIG. 7, panel B) had lower levels of nonspecific binding,lower backgrounds, and higher signals from fluorescent bacteria bound tothe peptide spots than avidin-coated slides patterned with C-terminalbiotin-magainin (FIG. 7, panel A, P <0.05). Since the calculations fordetection limits were based on both specific and nonspecific binding andvariability thereof, both E. coli and S. typhimurium could be detectedat significantly lower levels where magainin I had been immobilizeddirectly (FIG. 8). Detection limits for labelled E. coli and S.typhimurium on covalently immobilized magainin I were 1.6×10⁵ cell/mLand 6.5×10⁴ cell/mL, respectively; detection limits on surfaces wheremagainin I was immobilized via its C-terminal biotin were at least4-fold higher −6.8×10⁵ cell/mL and 5.6×10⁵ cell/mL, respectively.Although sensitivity for E. coli was an order of magnitude poorer inmagainin-based assays than analogous, optimized antibody-based assays(L. Shriver-Lake, personal communication), the LOD for Salmonella was inthe same range as determined previously with the antibody used here as acontrol.

The ability to capture target bacteria was strongly dependent on thedensity of immobilized magainin on the sensor surface (FIG. 9).Bacterial binding to control antibodies saturated at approximately 60 nMantibody in the patterning solution, independent of whetherimmobilization was direct or via avidin-biotin interactions. However,the AMP-based assays required significantly higher concentrations ofmagainin in solution during the immobilization step before saturationwas observed (0.4 mM); this effect was observed with both C-terminalbiotinylated magainin I and magainin immobilized covalently. Thisdifference in concentrations required to achieve optimal surface densitywas not explained by the 80-fold difference in molecular size.

Although bacterial binding was demonstrated to magainin I immobilizeddirectly and via a C-terminal biotin, no binding of either labeledspecies was observed to immobilized magainin biotinylated using anamine-specific biotin. As the initial interaction of α-helical AMPs withmembranes of target bacteria is postulated to occur through binding ofpositively charged amino acids on the AMP with negatively chargedphospholipids in the bacterial membrane, the lack of binding activityobserved in these studies may well have been due to modification of anamine-containing residue critical to this initial process. Thispostulate was supported by the ability of magainin with a C-terminalbiotin to bind cells, albeit at a lower level than magainin immobilizeddirectly. The potential for modification of an essential amine moietywas further exacerbated by modification of multiple residues by theamine-specific biotin. In spite of the 1:1 molar ratio (biotin:magaininI) used in the labeling reaction, peptides with molecular weightscorresponding to incorporation of one, two, and three biotins wereobserved through electrospray mass spectrometry. A similar over-labelingphenomenon has also been observed with the polymyxin family of AMPs,with consequent loss of microbial binding activity.

This study showed that characteristics of surface chemistry commonlyconsidered as disadvantages in other systems (e.g., lack of diffusionand steric hindrance) worked to advantage when immobilizing a smallpeptide for detection of bacterial species. As the majority of aminemoieties targeted by the cross-linker reside in the amino-terminaldomain of magainin, the domain presumed responsible for the initialinteraction with microbial membranes, a decrease in binding activity ofthe immobilized species (versus unmodified and free in solution) was notunexpected; furthermore, as others have shown that net charge onmagainin greatly affect its activity, modification of these chargedresidues was also assumed to adversely affect binding activity.Therefore, it was surprising that magainin I immobilized via itsC-terminal biotin (with native +4 charge) did not bind bacterial cellsas well as magainin immobilized directly using an amine-specificcrosslinker. It is believed that steric hindrance encountered during thedirect immobilization procedure may have prevented modification ofresidues essential for target binding, as well as prevented modificationof multiple sites. Such over-labeling was observed when magainin I wasreacted (in solution) with an amine-reactive biotin; a similarphenomenon was observed with other amine-rich AMPs. Moreover, it ispossible that the orientation of the directly immobilized magainin isoptimal for target binding. It is not determined which amino acidresidues are directly linked to the surface.

Furthermore, the binding activity of magainin may have been improved bythe higher surface density when immobilized directly. Binding of labeledcells was observed when high concentrations of magainin were immobilizedonto surfaces through direct covalent attachment or via a biotin moietyon the C-terminal amino acid. The two-fold higher potential packingdensity of surfaces with directly immobilized magainin immobilizeddirectly versus magainin immobilized via its C-terminal biotin (assuminghelical conformation for magainin and 20-30 Å between biotin bindingsites on avidin), may have endowed these surfaces with sufficientavidity to detect bacterial targets at lower concentrations. Inaddition, given the high concentrations of magainin (˜0.4 mM) requiredfor optimal binding activity, formation of peptide multilayers wasprobable for both surfaces. However, it is possible that theconformation and/or orientation of magainin molecules immobilizeddirectly more effectively promoted formation of peptide multilayers.Peptide-peptide interactions have been postulated to be required forstrong target binding and microbicidal activity.

To date, there have been limited reports describing use of individualAMPs for capture and detection of target analytes. James et al.,“Detection of Endotoxin Using an Evanescent Wave Fiber-Optic Biosensor,”Appl. Biochem. Biotechnol., 60(3), 189-202 (1996) describe used ofpolymyxin B as a capture molecule on a fiber optic biosensor fordetection and quantification of E. coli lipopolysaccharide (LPS) in5-minute competitive assays. The detection limit in these polymyxinB-based assays, approx. 10 ng/mL, calculates to approximately the samenumber of bacteria per mL (3×10⁵-1.3×10⁶ cells/mL) as observed withmagainin I in these studies, assuming LPS monomer molecular weight ofLPS between 4 and 20 kDa, and 1.2×10⁶ LPS molecules per cell; magaininhas also been observed to bind to LPS. A report has recently beenpublished describing use of cecropin P1, another amphipathic α-helicalAMP, to immobilize E. coli cells onto microtitre plates. (Gregory et al,“Immobilization of Escherichia coli cells by use of the antimicrobialpeptide cecropin P1,” Appl. Environ. Microbiol., 71(3), 1130-1134(2005).) As the thrust of this latter study was bacterial enrichment,the total analysis time (2.5 hours) and detection limits (˜10⁷ cfu/mL)were significantly different from those obtained in the present study.

EXAMPLE 2

Luminex—“Sandwich”-type Luminex assays had been developed, usingantibodies for target capture (initial recognition) and AMPs fordetection of bound targets (FIG. 10, format A). Development ofAMP-capture assays for Luminex (FIG. 10, format B) has begun; theseresults from Luminex should be more comparable to those from the ArrayBiosensor for which AMP-capture assays have been developed. To date,this system has successfully shown capture of E. coli, Salmonella, andbot toxoid A using AMP-derivatized Luminex beads (FIG. 11). AlthoughLODs for E. coli and Salmonella were the same as those obtained inantibody-based Luminex assays (10²-10³ on bactenecin and 10⁵ cfu/ml onPMB, respectively), bot toxoid A could be detected only at extremelyhigh concentrations (1 μg/ml); these latter results contrast greatlywith results obtained using the Array Biosensor. Neither Y pestis norCoxiella could be detected at the concentrations tested on Luminex usingAMP-coated Luminex beads for capture.

These AMP capture assays have been modified to include AMP-NR beads astracers (FIG. 10, format C). However, large increases in backgroundsignals have been observed in these AMP-AMP assays, due to the AMP-NRbeads binding non-specifically to AMP-derivatized Luminex beads; thehigh background signals have to date prevented measurement of anysignificant signals above background values. In spite of thesepreliminary results, it is anticipated that use of additional blockers,chaotropes, and/or higher salt concentrations will lead to improvementsin the AMP-AMP assays. Furthermore, it is believed that generic use ofAMP-derivatized beads (Nile Red beads, magnetic beads) may lead tofurther improvements in both Luminex and Array Biosensor.

EXAMPLE 3

Binding patterns—Above, differences in binding between E. coli andSalmonella, two very closely related species, using AMP tracer beads inLuminex assays are demonstrated. This was extended to include additionalspecies on Luminex using AMP tracers (FIG. 12) and as well as AMP-Lxbeads for capture (FIG. 13). Similar results have been observed in theArray Biosensor (FIG. 14). Several particularly noteworthy observationshave been made with regard to binding patterns. Bot toxoids A and B,antigenically similar proteins, bind to different AMPs. The bindingaffinities for melittin in the assays run contrary to the hypothesisthat inhibition of bot toxins by various AMPs is most likely caused byAMP binding to active sites. Melittin contains the KR sequence requiredfor cleavage by bot toxin A peptidase activity, but is boundpreferentially by toxoid B; conversely, cecropin A is boundpreferentially by toxoid A, but does not contain any of the sequencesrequired for cleavage by toxin A peptidase activity. Another interestingobservation was that killed E. coli O157:H7 bound to a different subsetof AMPs than a non-pathogenic strain, ATCC 35218; a similar differencein affinity for cecropin P1 between E. coli O157:H7 and a non-pathogenicstrain was noted by Gregory, Id.

The effects of contaminating species on detection and identification oftarget species have been assessed. Specifically, an excess of E. coliO157:H7 was spiked into solutions of bot toxoid B and analyzed in ArrayBiosensor arrays, using antibody directed against botulinum toxin as atracer; therefore, only binding of bot toxoid B would be detected.Although a small decrease in binding activity was observed, thisdifference was not statistically significant and the limit of detectionon melittin (≦5 ng/ml) was not affected. However, at present, the onlydata analysis used for pattern recognition is normalization to a singleAMP. This methodology does not yet take into account differences inbinding due to varying target concentrations.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that the claimed invention may be practiced otherwise than asspecifically described. Any reference to claim elements in the singular,e.g., using the articles “a,” “an,” “the,” or “said” is not construed aslimiting the element to the singular.

What is claimed is:
 1. A device comprising: one or more substrates; anda plurality of capture species independently selected from the groupconsisting of antimicrobial peptides, cytotoxic peptides, antibiotics,and combinations thereof; wherein the capture species are bound to thesubstrates; wherein at least two of the capture species are capable ofmulti-specific binding to one or more biological targets.
 2. The deviceof claim 1, wherein at least two of the capture species have overlappingbut not identical affinity properties.
 3. The device of claim 1, whereina majority of the capture species are capable of multi-specific binding.4. The device of claim 1, wherein the device is a biosensor.
 5. Thedevice of claim 1, wherein the biological target is selected from thegroup consisting of bacteria, fungi, viruses, rickettsiae, toxins, andcombinations thereof.
 6. The device of claim 1, wherein the biologicaltarget is a toxin.
 7. The device of claim 1, wherein the capture speciesare selected from the group consisting of alamethicin, peptaibols,apidaecin, bacitracin, bactenecins, bombinin, brevinin, buforins,cathelicidins, cecropins, cepaphalosporins, cytolysins, dermaseptins,defensins, esculentins, gramicidins, hemolysins, histatin, indolicidins,beta-lactams, lactoferricin, nisin, lantibiotics, magainins,mastoparans, melittin, moricin, parasin, pediocin, penicillins,polymyxins, protegrins, ranalexin, streptogamins, tachyplesins,teichoplanin, thionins, vancomycin, vibriolysins, derivatives thereof,and combinations thereof.
 8. The device of claim 1, wherein the capturespecies include at least one naturally occurring peptide.
 9. The deviceof claim 1; wherein the substrate comprises a surface functionalizedwith a crosslinker; and wherein the capture species are covalently boundto the crosslinker.
 10. The device of claim 1, wherein the capturespecies are non-covalently bound to the substrate.
 11. The device ofclaim 1; wherein the capture species is bound to a protein or scaffold;and wherein the protein or scaffold is covalently or noncovalently boundto the substrate.
 12. The device of claim 1; wherein the substrates area plurality of microspheres; and wherein the separate subsets ofmicrospheres comprise different capture species or combinations thereof,the microspheres of each subset encoded by a different ratio of twodyes.
 13. The device of claim 1, further comprising: a source of one ormore tracer species comprising a recognition element capable of bindingto the at least one biological targets and a signal generating element.14. The device of claim 13, wherein the signal generating elementisselected from the group consisting of fluorophores, chromophores,fluorophore-labeled species, chromophore-labeled species, fluorescentnanospheres or microspheres, an enzyme or catalyst capable of producingan opto-electronic signal, and fluorescent nanospheres or microspherescoated with one of the capture species.
 15. The device of claim 14,wherein the fluorophore-label is selected from the group consisting ofCy3, CyS, cyanine dyes, phycobili proteins, and fluorescent protein. 16.The device of claim 1, further comprising: a detector capable ofindependently detecting the presence of the tracer species or thebiological target bound to each capture species to generate a bindingpattern.
 17. The device of claim 16, further comprising: a database ofat least one of the biological targets characterizing the biologicaltarget by its relative binding affinity for at least one of the capturespecies; and a system for performing a pattern recognition algorithm toidentify the biological target based on the binding pattern.
 18. Thedevice of claim 1, further comprising: a detector selected from thegroup consisting of opto-electronic, surface plasmon resonance,interferometry, and quartz crystalline microbalance.